LC-MSN METHOD FOR QUALITATIVE & QUANTITATIVE ANALYSIS OF COMPLEX LIPID MIXTURES

LC-MSN 定性方法

• 批准号: 8170855
• 负责人: Catherine E. Costello
• 金额: $ 0.39万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8170855
• 关键词: Biological; Chromatography; Complex; Computer Retrieval of Information on Scientific Projects Database; Detection; Fractionation; Funding; Genetic; Glycolipids; Grant; Human Milk; Institution; Investigation; Ions; Laboratories; Left; Length; Lipids; Literature; Low-Density Lipoproteins; Mass Spectrum Analysis; Methodology; Methods; Modification; Molecular; Persons; Phase; Phospholipids; Production; Proteins; Publishing; Qualitative Methods; Research; Research Personnel; Resources; Sampling; Scanning; Source; System; United States National Institutes of Health; base; interest; lipid disorder

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. While nanospray MS is a good choice for the characterization of simple lipid mixtures (1,2), it is often not sufficient for the qualitative and quantitative analysis of highly complex samples. Most separation methods described are limited, in that they either target only specific classes of interest (3), or are not well suited for MS, the superior detection method, especially for analyses of small amounts of samples. We have developed a simple, reproducible three-step method for lipid analysis by adapting separation systems described in the literature for the chromatography of lipids (4,5). After an optional initial fractionation, normal phase HPLC-MS first provides class separation and then a reversed phase LC-MS/MS system answers remaining questions. Methods: (a) Isolated and extracted LDL lipids and lipid standards are separated by 1D or 2D LCand are detected by mass spectrometry in positive and negative ion modes. Two different gradients are used for the separation of more nonpolar and more polar lipids. Quantification is based on this step. (c) Fractions obtained can be further characterized by LC-MS/MS using a triple quadrupole, QoTOF MS, or LTQ-Orbitrap MS, or by nanospray MS/MS and/or precursor ion scanning. Lipid and glycolipid standards containing diverse nonpolar, phospho- and glycolipids have been reproducibly separated on the basis of polarity. This step, when used for biological samples, also serves to protect the following column, but is not always necessary. The accuracy of the quantification depends mostly on the quality of internal and external standards available. The collected fractions are partially investigated by nanospray MS (MS/MS, precursor ion scanning and neutral loss scanning) for the determination of the molecular species present. A clean separation of molecular species can be achieved on a reversed phase column, especially with respect to the low abundant PEs. The LCMS methodology provides fairly robust and technically simple methods for the investigation of complex lipid mixtures. We have applied the methods to the analysis of lipids associated with full-length and truncated apolipiprotein in a normal indivicual and one who has a genetic modification that results in production of the truncated protein, and to lipids and glycolipids from other biological sources. We have published the results for LDL (7) and have drawn significant interest, judging from the number of investigators from the US and elsewhere who have contacted us about this approach as they begin to implement it in their own laboratories. Murphy et al.later published a modification to the method that simplifies the extraction and chromatography but leaves behind the phospholipids. (8) We are now investigating lipids from human milk and from persons with lipid disorders. 1) M. Puffer and R.C. Murphy (2003). Mass Spectrometry Reviews 22, 332-64. 2) X. Han and R.W. Gross (2005). Mass Spectrom Rev. 24, 367-412. 3) R.C. Murphy et al. (2001). Chem. Rev. 101, 479-526. 4) J. Hamilton, and K. Comai (1988). Lipids 23, 1046-49 & 1150-53. 5) W.W. Christie et al. (1995). J. High Resol. Chromatogr. 18, 97-100. 6) F.K. Welty et al. (1991). J. Clin. Invest. 87, 1748-1754. 7) U. Sommer et al. (2006) J. Lipid Res. 47, 804-814. 8) P. M. Hutchins et al. (2008) J. Lipid Res. 49, 804-813.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 虽然nanospray MS是表征简单脂质混合物的良好选择（1，2），但它通常不足以用于高度复杂样品的定性和定量分析。所描述的大多数分离方法都是有限的，因为它们要么仅针对感兴趣的特定类别（3），要么不太适合MS（上级检测方法），特别是对于少量样品的分析。我们已经开发了一种简单的，可重复的三步法脂质分析，采用文献中描述的脂质色谱分离系统（4，5）。在可选的初始分馏后，正相HPLC-MS首先提供类别分离，然后反相LC-MS/MS系统回答剩余的问题。 研究方法：（a）分离和提取的LDL脂质和脂质标准品通过1D或2D LC分离，并通过正离子和负离子模式下的质谱法进行检测。两种不同的梯度用于分离非极性较大的脂质和极性较大的脂质。定量基于此步骤。(c)所获得的级分可以通过使用三重四极杆的LC-MS/MS、QoTOF MS或LTQ-Orbitrap MS或通过纳米喷雾MS/MS和/或前体离子扫描来进一步表征。 含有不同非极性、磷脂和糖脂的脂质和糖脂标准品已根据极性进行了可重复分离。当用于生物样品时，该步骤也用于保护随后的柱，但并不总是必要的。定量的准确性主要取决于可用的内部和外部标准的质量。 通过纳米喷雾MS（MS/MS，前体离子扫描和中性损失扫描）对收集的馏分进行部分研究，以确定存在的分子种类。可以在反相柱上实现分子种类的清洁分离，特别是对于低丰度PE。 LCMS方法提供了相当稳健和技术上简单的方法， 复杂脂质混合物的研究。 我们已经应用的方法来分析脂质与全长和截短的载脂蛋白在一个正常indivicual和一个谁有一个基因修饰，导致生产的截短的蛋白质，并从其他生物来源的脂质和糖脂。我们已经发表了LDL（7）的结果，并引起了极大的兴趣，从美国和其他地方的研究人员的数量来看，他们开始在自己的实验室实施这种方法。Murphy等人后来发表了对该方法的修改，简化了提取和色谱，但留下了磷脂。(8)我们现在正在研究来自人乳和脂质紊乱患者的脂质。 1)M. Puffer和R.C. Murphy（2003年）。Mass Spectrometry Reviews 22，332-64. 2)X. Han和R.W.毛额（2005年）。Mass Spectrom Rev. 24，367-412. 3)R.C. Murphy等人（2001年）。101，479-526。 4)汉密尔顿和K. Comai（1988年）。Lipids 23，1046-49 & 1150-53. 5)W.W. Christie等人（1995年）。高解析度J. High Resol. Chromatogr. 18，97-100。 6)法光Welty等人（1991年）。J. Clin. Invest. 87，1748-1754。 7)联合Sommer等人（2006）J. Lipid Res.47，804-814. 8)P. M. Hutchins等人（2008）J. Lipid Res.49，804-813.